1. Field of the Invention
The present invention relates to a reaction site array having plural reaction sites, the preparation process thereof, the reaction process using the reaction site array and a quantitative determination method of a substance in a sample solution using the reaction site array, for the use of screening of chemicals such as curative medicines, of gene fingerprinting, of gene sequencing by hybridization (SBH: Sequencing By Hybridization) and of simultaneous multi-detection of subject materials, which can be used for so-called combinatorial chemistry where plural reactions are carried out in a trace amount at the same time.
2. Related Background Art
Recently, so-called combinatorial chemistry has been attracting attention, in which, for example, a plurality of oligopeptides expected to interact with the target medicine are prepared, and the interaction between the oligopeptides and the various chemicals to be screened are analyzed, to identify the target medicine. Because the approach of random drug design is inefficient, and the evaluation of the designed and synthesized drugs with animal tests etc. is time consuming and expensive, combinatorial chemistry is now required as an alternative measure.
As probes for such a combinatorial chemistry, there are above-mentioned oligopeptides. As a means to bind such probes onto the solid, latex particles having functional groups on their surfaces to bind the probe are commercially available (Calbiochemxe2x80x94Novobiochem Inc.). Further, U.S. Pat. No. 5,143,854 discloses a preparation method of an oligopeptide array using photolytic protecting groups and a photolithography process in combination.
In detecting target nucleic acids having a certain base sequence by using a nucleic acid probe, instead of conventional methods such as Southern hybridization, a method has been proposed where plural types of nucleic acid probes are immobilized onto a solid support and then test samples including target nucleic acids are hybridized thereto and the detection is conducted as in combinatorial chemistry.
For example, Japanese National Publication of PCT Application No. 3-505157 discloses an analytical device for polynucleotide sequence which comprises the entire or specific parts of a full set of oligonucleotides with a certain length immobilized onto a support. Further, in U.S. Pat. No. 5,202,231, a similar analytical method for sequencing by hybridization of polynucleotide is proposed. In U.S. Pat. No. 5,424,186, a preparation process of a nucleic acid probe array onto a solid support by a combination use of photolytic protecting groups and a photolithography process is disclosed.
In enzyme-immunoassay, generally, reaction is carried out on a microplate having a maximum of 96 wells and the results are read by a microplate reader for simultaneous multi-item or multi-sample reaction and detection. This method has a limitation in high throughput analysis of a large number of samples.
One of the main concerns of combinatorial chemistry is how to supply various reaction species to a reaction site effectively; in other words, how to supply effectively a variety of reaction species each in a small amount (in a small liquid volume) to a reaction site without cross-contamination. From this viewpoint, the microplate method described above has theoretical limitations, although efficient and throughput systems have been developed recently using robot technology. It has another problem that a relatively large volume of liquid, i.e., from about 20 xcexcl to about 100 xcexcl, is necessary to be supplied to one well.
Also in the synthetic method of a probe array on a solid phase using a photolytic protection group and photolithography described in the above-mentioned U.S. Pat. Nos. 5,143,854 and 5,424,186, although it is possible to array a variety of probes onto a support, each probe lies on the same plane so that substances to be reacted with the probe are supplied to the entire probes, making it impossible to conduct different reactions with each probe. In addition, as the probes, oligopeptides or oligonucleotides which are synthesized on a solid support are used without any purifiying treatment, and thus it is not possible to confirm whether desired probes are synthesized, and by-products which are inevitably synthesized during the probe synthesizing steps such as oligomers shorter than the probes etc. cannot be removed.
As a means to solve these problems, there has been proposed to supply reaction species already synthesized and purified to the reaction site using a microdispenser. For example, Khrapko et al. introduce a method to make a DNA probe array by spotting a DNA solution using a micropipet onto a polyacrylamide gel (J. DNA Sequencing and Mapping, 1, 375-388, 1991). According to this method, a DNA solution of a relatively small amount can is fed, but the region to which the DNA solution is fed cannot be specified, thus causing a problem in quantitation. Also, cross-contamination between the juxtaposed spots may occur when probe solutions are fed. The same problem may arise when the other reaction species are fed.
There has been also proposed a method for stepwise synthesis of nucleic acid probes performed on mainly porous solid matter, where the ink-jet method is employed to reduce the feeding amounts of reaction species and to achieve reactions in various kind and a large number (International Publication of PCT Applicatin No. WO95/25116). This method has problems in stepwise synthesis of probes and in the not-specified feeding regions.
There have been proposed some measures to solve the problem that the regions for the reaction species can not be specified.
Chrisey et al., for example, introduce a method where a silane coupling agent having appropriate functional groups is applied onto a support and subjected to patterning, then onto which DNA probe solutions are spotted to prepare a DNA probe array (Nucleic Acid Research, Vol.24, Number 15, 3040-3047, 1996).
Lemmo et al. introduce a method to feed reagents using a microdispenser into each well of a polypropyrene sheet (plate) molded to have a large number of wells on its surface (Anal. Chem., 69, 543-551, 1997). Specifically, a reagent solution of about 8 xcexcl each is fed with a microdispenser into each well of a polypropyrene resin plate having 48xc3x9748 wells. The well size is presumed to be 3 mm in diameter and 2 mm in depth, and the size of the molded plate is described to be 8.5 inchesxc3x9711 inches. With molding, the feasible well size is about several millimeters as in the above mentioned case, and when the whole plate size is considered, the number of wells composing the array is not more than 48xc3x9748, and the entire plate size is not so small. When actually used in combinatorial chemistry, many more kinds of probe species are desirably used and a much smaller plate is desirable. In addition, since a plate made of polypropylene is water repellent, it is difficult to distribute aqueous solutions of biomaterials such as nucleic acid into small wells, and undesirable cross contamination may occur between the adjacent wells.
Japanese National Publication of PCT Application No. 7-508831 discloses a method to supply a nucleic acid probe solution using a microdispenser to patterned regions of a silicone support. According to this method, both the probe species number and array size seem to fill the requirement, but there still remains the problem of cross contamination when probes are fed or when test samples are applied.
According to the method disclosed in International Publication of PCT Application No. WO95/35505, a nitrocellulose filter backed with a non water-permeable film is sectioned with silicone rubber and then to these sections a DNA solution is supplied to form a DNA array by non-covalent bonding. There is disclosed a method to examine plural samples at the same time without cross contamination by providing plural sets of sections divided with silicone rubber on the support, but not specifically about individual DNA reaction regions.
According to one aspect of the present invention, there provided is a process for producing a reaction site array which comprises a plurality of reaction sites to conduct a reaction between two or more kinds of substances in a liquid medium, each of the reaction sites being composed of a first region having a first affinity to the liquid medium and separated from each other by a second region having a second affinity to the liquid medium which is lower than the first affinity, and the second region being raised from the first region, the process comprising the steps of:
providing a support; and
forming a matrix pattern having the second affinity and raised from the support surface, to form the first region composed of the support surface exposed corresponding to the matrix pattern and the second region composed of the matrix pattern.
According to another aspect of the present invention, there provided is a reaction site array comprising a plurality of reaction sites to conduct a reaction between two or more kinds of substances in a liquid medium, wherein each of the reaction sites is composed of a first region having a first affinity to the liquid medium and separated from each other by a second region having a second affinity to the liquid medium which is lower than the first affinity, and the second region is raised from the first region.
According to the further aspect of the present invention, there provided is a process for conducting a reaction between two or more kinds of substances in a liquid medium comprising the steps of:
providing a reaction site array comprising a plurality of reaction sites being composed of a first region having a first affinity to the liquid medium and separated from each other by a second region having a second affinity to the liquid medium which is lower than the first affinity, and the second region being raised from the first region, and
applying the substances to at least one of the reaction sites and reacting the substances in the sites.
According to the further aspect of the present invention, there provided is a process for quantifying a first substance contained in a sample liquid comprising the steps of:
a) providing a reaction site array comprising a plurality of reaction sites, each of the reaction sites being composed of a first region having a first affinity to the sample liquid and separated from each other by a second region having a second affinity to the sample liquid which is lower than the first affinity, and the second region being raised from the first region;
b) supplying the sample liquid to the reaction site;
c) supplying to the reaction site a reagent providing a detectable and quantifiable signal when interacting with the first substance to enable the quantitative detection of the first substance; and
d) quantitatively detecting the signal.
According to the present invention, the reaction sites are wells formed on a substrate in a matrix-like pattern, and the bottom (the first region) of the well is the exposed substrate having a high affinity to the liquid medium and the surrounding wall (the second region) raised from the substrate is made of a material having a low affinity to the liquid medium. Such a constitution enables smooth feeding of the reaction solution comprised of the liquid medium and reaction substances, prevention of the solution from flowing over the raised region because of the low affinity of the surface of the raised part to the solution, that is, prevention of cross contamination between the adjacent wells. Due to smooth feeding of the solution to the wells, the solution can be fed almost several ten times as much as the volume of the well.
According to the present invention, the reaction site array having such functions can be effectively prepared with high accuracy.
A matrix pattern forming wells can be made using a fine patterning technology described hereinafter, a large number of sufficiently small reaction sites can be made on a chip of, for example, 1 cmxc3x971 cm.
In the present invention, that the support surface has an affinity to a liquid medium means, it has an affinity, in addition to the affinity to the liquid medium itself, to the liquid medium containing one or more substances such as reactants, auxiliaries required in reaction, reagents for quantitative or qualitative analysis, and reaction products. The same is said to the xe2x80x9cnon-affinityxe2x80x9d of the projecting part has no affinity.